A biopolymer specifically binding to a target substance or a low molecular compound targeting a biomolecule is expected as a medicinal drug candidate, which binds specifically to a target substance, thereby exhibiting an effective physiological activity in a living body. Such a biopolymer or a low molecular compound is also expected as a target-substance capturing molecule of a biosensor using a specific binding ability to a target substance as described above.
As an example of such a biopolymer, an antibody may be mentioned. Antibodies bind to various foreign substances invading into the body fluid of an animal by recognizing various structures on the surfaces of the foreign substances and detoxicate these substances by its immune system. In short, antibodies are one class of the proteins functioning in a self-defense mechanism. To function such a mechanism effectively, an antibody has a molecular diversity (that is, having a number of different amino acid sequences in order to bind to various foreign substances). The number of kinds of antibodies is estimated be 107 to 108 per individual animal. Since an antibody has such specific antigen recognition ability, high antigen binding ability and molecular diversity, it is expected as a medicinal drug candidate and a target-substance capturing molecule.
An antibody is a protein generally having 150 kDa consisting of two of two types of polypeptide chains; one is called a heavy chain of about 50 kDa, and the other is called a light chain of about 25 kDa.
The heavy chain and the light chain each have a variable region and a constant region. The light chain is a polypeptide chain constituted of two domains; one variable region (called a light chain variable region: VL) and one constant region (CL). On the other hand, the heavy chain is a polypeptide chain constituted of 4 domains, that is, a single variable region (heavy chain variable region: VH) and three constant regions (CH1 to CH3). Each domain consists of about 110 amino acids and has a cylindrical structure, in which beta sheets are arranged in antiparallel and mutually connected via an S—S bond to form a very stable layer structure.
Antibody molecules characteristically have the binding diversification capable of binding to various types of antigens. The binding diversification is ascribed to the diversity in amino acid sequences of three complementarily determining regions (CDRs) having a loop structure and present in each of the variable regions (VH and VL).
The CDR is also called a hypervariable region. Each domain of the VH and VL has three CDRs. These CDRs are arranged on the surface of an antibody molecule and separated from each other by a region called a framework, which has a relatively common amino acid sequences between the VH and VL domains. The antibody recognizes a spatial arrangement of functional groups of a recognition site (antigenic determinant: epitope) of an object, a target substance. By virtue of this, the antibody can recognize a molecule highly specific. The presence of CDR contributes to forming a hypervariable loop structure of the antibody.
Antibodies can be produced by a method in which a desired antigenic substance is injected in combination with an adjuvant to an animal recipient (such as a rabbit, goat or mouse) at predetermined time intervals and antibodies present in the serum are recovered. Antibodies can be also produced by another method in which B cells capable of producing the antibodies are taken from the aforementioned animal recipient, fused with established tumor cells to prepare hybridoma cells, and then, the hybridoma cells are allowed to produce antibodies, followed by purifying the antibodies.
The antibodies produced by the former method contain various types of antibodies (a mixture of antibodies) recognizing different structures on the surface of the antigenic substance used in immunization. Such a serum containing a plurality of antibodies binding to a single antigen is called a polyclonal antibody. However, the antibodies produced by the latter method are called a monoclonal antibody. This is because since the antibody-producing B cells can produce only one type of antibody. The antibodies produced from one of the hybridoma cells mentioned above come to be single-type monoclonal antibodies.
In either method, an animal must be immunized by injecting a target substance, an antigen. Whether an antibody, that is, a capturing molecule capturing a desired target substance, is obtained or not cannot be confirmed until the antibodies or the serum is taken and its titer (avidity) is checked. In short, in either a polyclonal antibody or a monoclonal antibody, the characteristics of the antibody obtained vary depending upon the immune system of an animal to be immunized. Furthermore, even if the hybridoma cells capable of producing a monoclonal antibody exhibiting a binding ability to a target substance can be obtained, an efficient genetic engineering method has not yet been found for improving the binding ability of the obtained antibody, at present. Moreover, generally, production of an antibody against a target substance having an analogous structure to that of a bio-constituent of an animal recipient, such as a sugar and a lipid, cannot be expected even if it is a non-self substance. In other words, production of an antibody specifically binding to such a target substance cannot be expected in the immune system serving as a bio-defense system.
On the other hand, a combinatorial method is disclosed to obtain a capturing molecule binding to a target substance by using, for example, an antibody fragment containing at least a part of VH and VL, serving as a binding portion (such as Fab and a single chain Fv (scFv)) of an antibody to an antigen. In U.S. Pat. No. 5,969,108, there is a known technique that an antibody fragment as described above is fused with a phage, in particular, a coating protein of a fibrous phage, and used as a phage antibody having an antibody exposed on the surface. Such a phage having an antibody exposed on the surface of the coating protein is disclosed in not only U.S. Pat. No. 5,969,108 but also the pamphlet of International Publication WO 88/06630, WO 9215606, which discloses that the phage is used in a method of selecting a clone of an antibody fragment. According to these methods, a clone capable of binding a target substance can be easily obtained compared to a conventional immunization method for obtaining an antibody. In short, a conventional method for producing antibodies, which is said to be difficult to express other than in animal cells, can be improved by cleaving an antibody into fragments to lower the molecule weight.
In an antibody display method represented by the aforementioned method, first, a Lead antibody fragment binding to a target substance is obtained under a specific selection pressure and mutated by a bioengineering approach. Then, a binding/selection experiment is repeatedly performed. As a result, an antibody fragment having a higher binding ability to the target substance can be obtained. These antibody display methods have a characteristic feature in that since the complicated immune system of a living body is not used to obtain an antibody to be bound to a target substance, it does not a matter whether an antigen is self-derived or nonself-derived. Furthermore, if a gene portion encoding the CDR portion of an antibody fragment is chemically synthesized, the size of a gene library also can be enlarged.
Furthermore, U.S. Pat. No. 5,910,573 discloses a capturing molecule formed of a monomer protein with which an amphipathic helix peptide capable of forming a dimer with scFV is fused. According to the description of the invention set forth in the publication, the invention has an advantage in that amphipathic helix peptides are interacted to form a dimer, which functions as a bivalent antibody. The publication also discloses a technique regarding a bivalent specific antibody in which scFv recognizing a different antigen is fused with the helix peptide chain. However, scFv has two disulphide bonds in the polypeptide chain. The disulphide bonds are likely to serve as an obstacle to inducing folding in a correct manner when the protein is synthesized or secreted. Therefore, a problem still remains in productivity.
In J. Mol. Biol., 2001, 312, 221-228, the amphipathic helix peptide is fused with each of the heavy chain variable region (VH) and the light chain variable region (VL) constituting Fv to form a protein. According to the disclosure, unlike the case of U.S. Pat. No. 5,910,573, the polypeptide chain constituting the protein contains only a VH or VL domain. By this method, it is expected to overcome the problem of productivity as mentioned above. However, a single capturing molecule disclosed in the publication alone binds only to a single epitope. Therefore, it is difficult to impart a binding specificity superior to an antibody and antibody fragment known in the art.
J. Mol. Biol., 1995, 246, 367-373 discloses improving the binding ability of a capturing molecule to a target substance HEL. To be more specifically, the document discloses that a single chain scFv (derived from D1.3 and HyHEL10) capable of binding to HEL is genetically fused to obtain a single stranded scFv dimer, which shows improved binding ability to HEL. Similarly, International Publication WO 2004/003019 pamphlet also suggests a technology regarding a target substance-capturing molecule that recognizes two different epitopes present on the surface of the same single target substance molecule; unfortunately, it fails to mention specific techniques.
However, even in the antibody and antibody molecule obtained by such a combinatorial method and genetic engineering method, it is still difficult to obtain a clone having an excellent binding ability to a substance such as a sugar and a lipid, at present.
On the other hand, it has been suggested that the in vivo behavior of a lipid and post-translational modification of a protein have biologically significant meanings. Therefore, it is expected to apply a capturing molecule, which has a high binding ability to a substance such as a sugar and a lipid, to not only biochemical/medical fields but also wide variety of fields.
However, when a protein constituted of a recombinant single stranded scFv as mentioned above is produced in a low yield (several mg/L in Escherichia coli), the molecular stability of the protein tends to be low. The antibody fragments, in particular, VH and VL fragments, have a hydrophobic site at which these fragments are associated with each other. The VH and VL fragments are associated with other via the hydrophobic site to form a hetero dimer (Fv), which is stable as a molecule. However, in a folding step of a protein after protein synthesis or during the protein secretion from a cellular membrane, the hydrophobic site of the antibody fragment domain, if it is present alone, may trigger aggregation of protein or the like. In addition, it is known that the antibody fragment has a lower stability also as a molecule, compared to an antibody. This is because the antibody fragment is a recombinant protein, which is prepared by deleting a constant region thereof in order to give top priority to a binding ability to a target substance, and which has a small area between polypeptide (molecule) chains. These problems of productivity and molecular stability may be obstacles to industrial application. Technical problems still remain.